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. 2024 Aug 21;108(1):445. doi: 10.1007/s00253-024-13276-2

Strategies and procedures to generate chimeric DNA polymerases for improved applications

Zhuoxuan Yu 1, Jufang Wang 1,2,✉
PMCID: PMC11339088  PMID: 39167106

Abstract

Abstract

Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation.

Key points

• Chimeric DNA polymerase is generated by rational design method.

• Strategies include domain exchange and addition of proteins, domains, and motifs.

• Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions.

Keywords: Chimeric enzyme, DNA polymerase, Domain exchange, Rational design, Thermal stability, Processivity

Introduction

As an essential component in DNA replication, DNA polymerase catalyzes the synthesis of a complementary DNA strand by adding nucleotides to the template strand. Since the discovery of DNA polymerase I capable of synthesizing DNA in vitro, and the unraveling of the complex mechanisms involved in DNA replication, we have been deepening our understanding of how genetic information is faithfully transmitted to the next generations (Lisova et al. 2022; Kuznetsova et al. 2022). This enhanced knowledge has laid the foundation for the development of numerous applications, including the polymerase chain reaction (PCR) (Mullis et al. 1986; Bhadra et al. 2020).

DNA polymerases, found in a wide ranges of organisms from eukaryotes and prokaryotes to viruses, were classified into seven families: A, B, C, D, X, Y and RT. Amazingly, enormous types of DNA polymerases in all seven families are rather conserved in structures, often described as a right-handed fold. This configuration includes distinct domains: the palm, which contains the polymerase’s active site; the thumb, responsible for binding the replicated DNA; and the fingers, which interact with the incoming nucleotides (Kuznetsova et al. 2022). In addition to the right-hand-like structural configuration, some DNA polymerases also possess additional domains such as the 3’ to 5’ exonuclease and/or 5’ to 3’ exonuclease domain (Zahurancik and Suo 2020; Dangerfield and Johnson 2023). Each domain within the DNA polymerase contributes uniquely to the enzyme’s specific functional characteristics.

Currently, DNA polymerase is widely used in biotechnological fields, including DNA amplification, sequencing, and molecular diagnostics (Ordóñez and Redrejo-Rodríguez 2023; Akram et al. 2023; Chen et al. 2016). Nevertheless, wild-type polymerase fails to meet the growing demand of DNA replication under harsh conditions, such as extreme temperature and high salinity, or replicating templates with low concentration or from ancient fossil samples (Wang et al. 2022; Takahashi et al. 2018). Thus, there is an urgent need to generate engineered DNA polymerases with improved precision and efficiency to address those demands and to further maximize the potential of this promising protein.

A lot of engineering strategies have been applied to improve the performance of DNA polymerase, and many of them proved to be successful (Nikoomanzar et al. 2020; Kuznetsova and Kuznetsov 2023). One of the most effective strategies is directed evolution, a powerful technique to generate polymerases with desired properties or improvements. Numerous genetic mutants were obtained through mutagenesis, error-prone PCR, or DNA shuffling, followed by a screening process to identify variants with desired characteristics. The selected variants were subjected to further iterative cycles, gradually enhancing the desired characteristics such as polymerase activity (Ma et al. 2016; Ahmad et al. 2023), broadening the substrate spectrum (Dangerfield et al. 2022), and improving incorporation efficacy (Wang et al. 2017). This method links genotype and phenotype without revealing the underlying mechanism, facilitating the creation of active variants with beneficial gain-of-function mutations.

Another significant strategy is rational design. Previous studies have uncovered the structure of DNA polymerases and the role of different domains within a polymerase (Kuznetsova et al. 2022). It provided detailed information about which site may be critical for conformation changes or catalytic activity. Rational design built a solid foundation for achieving the desired properties or applications such as the incorporation of modified nucleotide (Gardner et al. 2019; Wang et al. 2022), exonuclease silencing (Motré et al. 2008), and PCR (Bhadra et al. 2020).

Recently, some events of progress have been made by integrating specific heterologous regions or domains to create chimeric DNA polymerases, which is one of the rational design strategies. These chimeric polymerases can exhibit advantages from both of the parent polymerases, or enhanced specific functions to compensate for their drawbacks.

This review provides a concise overview of the latest developments in chimeric DNA polymerases for diverse applications, categorized based on their fusion strategies. It outlines the key strategies and general procedures used for creating these polymerases, as well as their potential for improved performance in various applications.

Current progress of generating chimeric DNA polymerases

Typically, to generate chimeric DNA polymerases with improved functions, amino acid sequences from two or more distinct proteins are attached together to form an integral polymerase, which exhibits a notable improvement of some enzymatic characteristics.

Over the past decades, detailed studies of crucial enzymatic characteristics have been carried out to comprehensively evaluate the effectiveness of the modified DNA polymerase. Moreover, an in-depth analysis of the polymerase structure and kinetics has revealed the domains associated with these enzymatic features, providing insights for rational design (Kuznetsova et al. 2022). The activity of the polymerase, generally referring to the rate of elongating new DNA strands from 5’ to 3’, is vital for DNA replication, usually defined as processivity, which is known as how much nucleotides can be added to the strand by polymerase per single binding event to the primers. It is also described as amplification efficiency when it comes to applications. For the polymerase itself, it is essential as well to have thermal stability and the capability to maintain amplification at specific temperatures (Śpibida et al. 2017) as DNA polymerase is widely applied in PCR and other isothermal amplification techniques. Many studies also focus on fidelity, which is the precision of base insertion, as well as inhibitor tolerance, indicating the capacity to maintain robustness and functionality against inhibitors such as dyes or ions in buffers. Additionally, considering the diverse potential application of DNA polymerase, characteristics like specificity, proofreading activity, and the ability to incorporate unnatural nucleotides are of significant interest (Table 1).

Table 1.

Description of enzymatic characteristics of interested DNA polymerases

Enzymatic properties Description Domain(s) related
Activity General rate of elongating new DNA strands from 5’ to 3’ Palm
Processivity Number of nucleotides added by polymerase per single binding event Palm, thumb, finger
Thermal stability Ability to resist high temperature without losing activity All
Fidelity Accuracy of base insertion Finger
Inhibitor tolerance Ability to remain functional against inhibitors (e.g., EDTA, heparin, Nacl or ions) All
Substrate specificity Ability to distinguish template with specific sequences or modification modifications Thumb, finger
Proofreading activity Activity of 3’ to 5’ exonuclease Exonuclease
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Improved characteristics or development of novel functions in chimeric DNA polymerases are achieved by the introduction of different domains or proteins to the original polymerase, including homologous domains of polymerases from various species, certain proteins for various functions like DNA binding, and particular patterns of protein structure.

Homologous domain exchange

DNA polymerases share a great similarity in palm domains with relatively conserved catalytic residues, while the thumb and finger domains may vary considerably (Braithwaite and Ito 1993). It provides extensive potential for polymerases to significantly improve certain characteristics or acquire novel functions without losing the original catalytic ability. A highly effective strategy is to conduct homologous domain exchange, wherein a specific domain of the native polymerase is replaced with a corresponding domain or a similar sequence from another polymerase.

There are many examples of generating chimeric bacterial DNA polymerases through homologous domain exchange. For instance, the segment from amino acids 292 to 423 in Taq polymerase was substituted with the sequence from amino acids 327 to 519 of E. coli pol I, which is also recognized as the 3’-5’ exonuclease domain of the Klenow fragment. This exchange endowed the Taq polymerase with both 3’-5’ exonuclease activity and proofreading capabilities (Villbrandt et al. 2000). A chimeric reverse transcriptase was constructed by fusing the 5’-3’ nuclease domain of T. Z05 DNA polymerase from Thermus species Z05 with the 3’-5’ exonuclease and polymerase domains of Tma polymerase from Thermotoga maritima. This enzyme could be utilized to perform RT-PCR for long templates in low concentration and quality instead of using a mixture of polymerase (Schönbrunner et al. 2006). Besides, a thermostable chimeric Tth-Taq DNA polymerase was constructed by fusing the N-terminal of Tth polymerase (amino acids 4 to 600) from Thermus thermophilus with the C-terminal of Taq DNA polymerase (amino acids 556 to 834). The resulting chimeric enzyme not only showed a fivefold increase in activity over Taq polymerase, but also displayed a much greater specificity in amplification compared to Tth polymerase, combining advantages from both original DNA polymerases (Ignatov et al. 2009). By replacing the finger and palm domains of KOD DNA polymerase from Thermococcus kodakarensis with those of Pfu DNA polymerase from Pyrococcus furiosus, the chimeric DNA polymerase exhibited processivity and thermal stability similar to those of KOD, while also possessing high fidelity derived from Pfu. The result indicated that the finger and palm domains could be the structural foundation for the fidelity of base insertion, while the thumb domain appears to be more critical for maintaining processivity (Faurholm et al. 2012).

In another case, helix N and helix P, which are critical regions of the nucleotide-binding sites in the RB69 DNA polymerase (gp43), were substituted with corresponding segments of DNA polymerase from the human cytomegalovirus (HCMV UL54). The chimeric gp43-UL54 polymerase exhibited increased sensitivity against foscarnet and acyclovir, facilitating further study to identify major determinants for the efficacy of these drugs (Tchesnokov et al. 2009).

A recent study has created a halophilic version of KOD DNA polymerase based on the latest finding about BR3 polymerase, which was explored from archaea that flourish in deep-sea brine environments. The exonuclease or thumb region of KOD was substituted with its homologous domain in BR3 (Fig. 1a), leading to a threefold enhancement in the chimera polymerase’s tolerance to NaCl, without compromising its thermal stability (Takahashi et al. 2018) (Fig. 1b).

Fig. 1.

Fig. 1

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Chimeric KOD DNA polymerase by homologous domain exchange (Takahashi et al. 2018). a Schematic of BR3 Pol, KOD Pol WT and chimeric polymerases by exchanging homologous domains between KOD Pol and BR3 Pol. The exonuclease and thumb domains of BR3 Pol are highlighted in blue and green, respectively. b The dissociation constants Kd (nM) of BR3 Pol, KOD Pol WT, KODBR3exo, and KODBR3thumb for binding to primer/template strands in buffers with either 100 mM or 300 mM NaCl. The maximum response units observed were fitted using the steady-state affinity model to calculate the equilibrium dissociation constant (Kd) for primer/template strands

The aforementioned examples serve as compelling evidence that domain exchange would be a highly effective strategy for the creation of novel characteristics in DNA polymerases (Table 2).

Table 2.

Progress of chimeric DNA polymerases by applying homologous domain exchange

Chimeric DNA polymerase Properties optimized/applications References
KOD + BR3 exonuclease Increased tolerance to NaCl (Takahashi et al. 2018)
KOD + BR3 thumb Increased tolerance to NaCl (Takahashi et al. 2018)
KOD + Pfu finger & palm Increased fidelity (Faurholm et al. 2012)
RB69 + HCMV UL54 helix P Obtained sensitivity to foscarnet and acyclovir (Tchesnokov et al. 2009)
RB69 + HCMV UL54 helix N Obtained sensitivity to foscarnet and acyclovir (Tchesnokov et al. 2009)
Taq + Klenow exonuclease Improved processivity, 3’–5’ exonuclease activity and proof-reading function (Villbrandt et al. 2000)
Tma + T. Z05 5′-nuclease Improved protein expression, RT-PCR (Schönbrunner et al. 2006)
Tth + Taq Improved activity and specificity of amplification (Ignatov et al. 2009)
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Integration of functional domain

Given the extensive sequence similarity among numerous DNA polymerases, a common strategy is to incorporate an extra domain with a specific function either at the terminus or in the middle of the polymerase sequence (Table 3), in addition to exchanging domains between polymerases.

Table 3.

Progress of chimeric DNA polymerases by integrating functional domain

Chimeric DNA polymerase Properties optimized/applications References
Klenow + T7 TBD Improved processivity (Bedford et al. 1997)
Taq + T3 TBD Improved processivity and fidelity (Davidson et al. 2003)
Sau-LF + T7 TBD Improved processivity (Zhai et al. 2019)
Stoffel + Pfu DBD Improved processivity and thermal stability, increased tolerance to lactoferrin and heparin, amplification efficiency of GC-rich template (Śpibida et al. 2018)
Bst-LF + Pab DBD Improved processivity and thermal stability, increased tolerance to NaCl, heparin, and ethanol, S. typhimurium detection (Li et al. 2023)
Gss + Pab DBD Improved processivity, whole genome amplification (Oscorbin et al. 2017)
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Many attempts have been done to improve the processivity or other enzymatic properties of DNA polymerases. A famous example of this approach involves the insertion of a 76-amino-acid-residue thioredoxin binding domain (TBD) derived from bacteriophage T7 DNA polymerase. This domain is inserted between the helices H and H1, located at the tip of the thumb domain of the Klenow DNA polymerase. In contrast to the original Klenow DNA polymerase, the chimeric Klenow-TBD displayed an approximately eightfold higher activity due to the presence of thioredoxin, while the activity of Klenow DNA polymerase itself stayed unchanged (Bedford et al. 1997). Another case also showed that the insertion of TBD from the T3 DNA polymerase enhanced the processivity and fidelity of Taq DNA polymerase (Davidson et al. 2003). Likewise, the addition of T7 DNA polymerase TBD adjacent to the N-terminus of Staphylococcus aureus DNA polymerase large fragment (Sau-LF) led to a more than twofold enhancement in processivity with the addition of thioredoxin. In contrast, the wild-type Sau-LF showed no significant enhancement (Zhai et al. 2019).

DNA-binding domain (DBD), another functional domain identified in many thermophilic organisms, has been extensively investigated and utilized for creation of chimeric DNA polymerase due to the notable binding affinity for both single-stranded and double-stranded DNA. In a particular application, the DBD from DNA ligase of Pyrococcus abyssi was fused with the N-terminus of Geobacillus sp. 777 DNA polymerase (Gss). This modification led to a threefold increase in processivity, which subsequently led to approximately a fourfold increase in the quantity of DNA product during whole genome amplification (Oscorbin et al. 2017). A recently developed hybrid enzyme, generated by fusing the identical DBD from Pyrococcus abyssi DNA ligase with the K431E variant of Bacillus stearothermophilus DNA polymerase large fragment (Bst-LF) (Fig. 2a), allows for real-time loop-mediated isothermal amplification (LAMP) at temperatures as high as 73 °C. This enzyme maintains its activity after exposure to 70 °C for 8 h (Li et al. 2023) (Fig. 2b and c). Additionally, fusion of DBD from Pyrococcus furiosus DNA ligase with Taq Stoffel fragment—a 62.7 kDa fragment of the thermostable Taq DNA polymerase—has demonstrated approximately a threefold increase in its processivity. This hybrid polymerase exhibits a significantly higher resistance to PCR inhibitors such as heparin or lactoferrin. Remarkably, it is capable of amplifying GC-rich DNA templates even as high as 78% GC contents (Śpibida et al. 2018).

Fig. 2.

Fig. 2

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Chimeric Bst DNA polymerase by integrating functional domain DBD (Li et al. 2023). a Schematic of Bst-LF WT, K431E variant of Bst-LF and chimeric polymerases by fusion with DBD from Pyrococcus abyssi DNA ligase at N-terminal or C-terminal of K431E. The lysine (K) and glutamate (E) at the 431 residue of Bst-LF and DBD domain are marked in blue, green, and red, respectively. b Thermal stability of Bst DNA polymerases variants. c Inhibitor tolerance of Bst DNA polymerase variants

Integration of functional protein

Enormous types of proteins within cells are vital for their survival and proper functioning, such as enzymatic catalysis, structural support, transportation, signaling, and regulation of gene expression (Morris et al. 2022). Beyond polymerases themselves, proteins that can help polymerase maintain stable formation, reduce clashes within domains, or facilitate polymerase-DNA interactions may be of great potential to improve the performance of current DNA polymerases.

Significant advancements have been achieved in improved processivity of DNA polymerase through the use of functional domains as previously outlined (Table 3). However, this approach is restricted to a limited number of polymerases since it highly depends on specific protein–protein interactions for maintaining the stability of the polymerase. Hence, there is a pressing need for an effective strategy for a broader range of polymerases. The incorporation of additional functional protein could offer a solution, as indicated by numerous studies (Table 4).

Table 4.

Progress of chimeric DNA polymerases by integrating functional protein

Chimeric DNA polymerase Properties optimized/applications References
Taq + Sso7d Improved processivity (Wang 2004)
Pfu + Sso7d Improved processivity (Wang 2004)
Stoffel + Sso7d Improved processivity (Wang 2004)
Sau-LF + Sso7d Improved processivity, increased tolerance to KAc (Wang 2004)
Tpa + Sso7d Improved processivity (Lee et al. 2010)
Gss + Sto7d Improved processivity, increased tolerance to NaCl, heparin, urea, and blood (Oscorbin et al. 2017)
M-MuLV RT + Sto7d Improved cDNA synthesis, increased tolerance to NaCl, heparin, formamide, and blood (Oscorbin et al. 2020)
RB69 + RB69 SSB Improved processivity (Sun et al. 2006)
Tzi + Sso SSB Improved processivity and fidelity (Lee et al. 2013)
Stoffel + Neq SSB-like protein Improved processivity and thermal stability, increased tolerance to lactoferrin, heparin, and blood (Olszewski et al. 2017)
Br512 + vHP47 Improved processivity and thermal stability (Paik et al. 2023)
Br512 variants + vHP47 Improved processivity and thermal stability, increased tolerance to urea, LAMP (Paik et al. 2022)
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DNA-binding protein Sso7d, originating from Sulfolobus solfataricus, is a monomeric, globular protein characterized by its surface rich in hydrophobic amino acids. It has drawn much attention due to its capability to bind to double-strand DNA in a sequence-independent manner. This characteristic implies that Sso7d could potentially provide a more broadly applicable approach to enhance the processivity of DNA polymerases by binding with non-specific DNA sequences (Guagliardi et al. 2002). The fusion of the heterologous Sso7d to Taq DNA polymerase, as depicted in Fig. 3a, has successfully increased its processivity without any negative effects on its catalytic activity or enzyme stability, thereby exhibiting significant benefits in PCR applications. The resulting chimeric enzymes, Sso7d-Taq and Pfu-Sso7d, displayed a ~ fivefold and ~ eightfold increase in processivity, respectively, while preserving comparable thermal stability (Wang 2004) (Fig. 3b). Furthermore, Sso7d has been successfully integrated with the Pfu polymerase and Taq Stoffel fragment (Wang 2004), as well as Tpa polymerase from Thermococcus pacificus and Sau-LF (Zhai et al. 2019), demonstrating its versatility in enhancing processivity for various DNA polymerases.

Fig. 3.

Fig. 3

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Chimeric Pfu, Taq, Gss DNA polymerase by integrating functional protein Sso7d and Sto7d (Wang 2004; Oscorbin et al. 2017). a Schematic of Pfu WT, Taq WT and their corresponding chimeric polymerases by adding Sso7d at N-terminal or C-terminal of the polymerase. The Sso7d protein is shown in green. b Processivity of Pfu and its derivatives of Taq-polymerase. c Schematic of Gss WT and its chimeric polymerases by adding Sto7d at N-terminal and C-terminal of polymerase with the His-tag. The His-tag and Sto7d protein are shown in blue and red, respectively. d Thermal stability and processivity of Gss-polymerase derivatives

A similar effect has been observed in the fusion of the Sso7d counterpart from Sulfolobus tokodaii, known as Sto7d protein, to Gss DNA polymerase (Oscorbin et al. 2017) (Figure 3c and d), and subsequently to Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Oscorbin et al. 2020). This chimeric reverse transcriptase, with Sto7d fused to its C-terminus, has shown a threefold increase in processivity during cDNA synthesis, as well as remarkable tolerance to various inhibitors, including NaCl, urea, formamide, and human whole blood (Oscorbin et al. 2020).

Potentials for using single-stranded DNA-binding protein (SSB), an auxiliary protein present at the replication fork, has garnered attention (Antony and Lohman 2019). SSB plays a crucial role in DNA replication by binding to single-stranded DNA, thereby preventing the formation of secondary structures and protecting ssDNA from exonuclease-induced degradation. Consequently, its strong DNA affinity could be beneficial for improving the DNA recognition capability of polymerases. In a study, the core domain of the C-terminus from the bacteriophage RB69 SSB was fused to the N-terminus of the corresponding DNA polymerase via a short, flexible linker Gly-Thr-Gly-Ser-Gly-Thr. The subsequent analysis revealed that this chimeric polymerase exhibited a substantial sevenfold improvement in processivity compared to the native RB69 DNA polymerase. This was evidenced by a significant increase in the average length of primer extension from 45.5 to 314.4 nucleotides, as well as maintaining the enzyme’s fidelity (Sun et al. 2006). In a subsequent study, SSB from Sulfolobus solfataricus was fused to the N-terminus of the Tzi DNA polymerase from Thermococcus zilligi by a linker composed of amino acids in the sequence Gly-Ser-Gly-Gly-Val-Asp. This modification led to a notable increase in both processivity and fidelity compared to the original Tzi DNA polymerase (Lee et al. 2013).

Some proteins similar to SSB have also been identified and employed in the creation of fusion proteins. A thermally stable protein from Nanoarchaeum equitans showed a surprising ability to bind to ssDNA, dsDNA, or mRNA without any structure-specific preference (Olszewski et al. 2015). This magical Neq SSB-like protein was soon integrated at the N-terminus of the Taq Stoffel fragment, leading to a significantly elevated extension rate and processivity. It also exhibited excellent thermal stability and tolerance to PCR inhibitors including lactoferrin, heparin, and whole blood, making this chimeric DNA polymerase a promising candidate for PCR applications (Olszewski et al. 2017).

The research for potential proteins that can be aligned with the existing polymerases has never been limited to the polymerase’s own components. One representative is the actin-binding protein villin, which plays a key role in the morphogenesis of microvilli, membrane protrusions of epithelial cells (Friederich et al. 1999). The C-terminus of the villin headpiece (vHP47) characterized by fast-folding kinetics due to strong hydrophobic interactions has been fused to polymerases; this fusion has shown improved amplification efficiency (Paik et al. 2022). Combined with sequence substitution predicted by machine learning, a comprehensive approach has been developed to generate polymerases that can withstand high temperatures. In this case, vHP47 was connected to several Br512 variants, and the resulting chimeric polymerase showed improved thermal stability, with a denaturation temperature up to 2.5 °C higher than the original Br512 variants. This enhancement allows isothermal amplification at 73 °C, a condition under which all commercially available Bst DNA polymerases are rendered inactive (Paik et al. 2023).

Integration of particular pattern of structure

Proteins function with precision and efficiency due to their specific three-dimensional configurations and conformations. These are determined by the organization of the helices, sheets, bulges, and residues within the protein’s structure, referred to as the primary and secondary structures. Any variations in these structures may lead to a drastic change in the properties, as many studies have confirmed (Sotomayor-Vivas et al. 2022). Therefore, adding a particular structural pattern to the polymerase based on existing knowledge could improve the performance of the DNA polymerase, and some progress in this area has been achieved, as indicated in Table 5.

Table 5.

Progress of chimeric DNA polymerases by integrating structural pattern

Chimeric DNA polymerase Properties optimized/applications References
Stoffel + Topo V HhH Improved processivity and thermal stability, increased tolerance to NaCl (Pavlov et al. 2002)
Pfu + Topo V HhH Improved processivity and thermal stability, increased tolerance to NaCl (Pavlov et al. 2002)
φ29 + Topo V HhH Improved processivity, B. subtilis genomic DNA detection (de Vega et al. 2010)
Bst-LF + Topo V HhH Improved processivity and thermal stability (Pavlov et al. 2012)
M-MuLV RT + 8xHis-Strep Improved cDNA synthesis and thermal stability (Yano et al. 2019)
φ29 + Topo V (HhH)2 Improved processivity, increased tolerance to KGlc, nanopore sequencing (Gao et al. 2021)
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Helix-hairpin-helix (HhH) motif is a common structural element that plays a role in sequence-non-specific DNA binding, as exemplified by human DNA polymerase β (Pelletier et al. 1996). These motifs are typically observed as single HhH or several (HhH)2 domains, such as DNA topoisomerase V (Topo V) which has been identified to contain 24 HhH motifs (Osterman and Mondragón 2022). Subsequent research has demonstrated that the HhH motifs of Topo V can improve the processivity of the Taq Stoffel fragment and Pfu DNA polymerase (Pavlov et al. 2002), φ29 DNA polymerase (de Vega et al. 2010), Bst-LF (Pavlov et al. 2012), etc. It was also found that the addition of different numbers of the HhH motif into catalytic domains of polymerase resulted in more or less an increase in thermal stability and inhibitor tolerance (Pavlov et al. 2012; Gao et al. 2021), as illustrated in Fig. 4. Even though HhHs motifs from different domains within the same polymerase shares a similar structure pattern, they showed a different effect on the thermal stability of the polymerase due to distinct partial charges at the same pH (Gao et al. 2021). Furthermore, chimeric φ29 DNA polymerase, which exhibits high salt tolerance t, has broadened its applications in genomic DNA detection (de Vega et al. 2010) and nanopore sequencing (Gao et al. 2021).

Fig. 4.

Fig. 4

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Chimeric Φ29 DNA polymerase by integrating structural pattern Helix-hairpin-Helix and its repeats (Gao et al. 2021). a Schematic of Φ29 WT and its chimeric polymerases by adding different copies of Helix-hairpin-Helix motif at C-terminal of the wild-type polymerase. The flexible Gly-Thr-Gly-Ser-Gly-Ala linker is shown in green. The Topo V (HhH)2 domain H (residues 696–751), H and I (residues 696–802), and E to L (residues 518–964) are shown in blue. b Processivity and tolerance of KGlc of Φ29 DNA polymerase and its derivative

Protein tags have been widely used for facilitating the purification of the recombinant expression of various proteins. In addition, some studies indicate that these tags could also improve the enzymatic characteristics of DNA polymerases. A His-Tag with 8 histidine residues and a Streptavidin Tag were fused to either the N- or C- terminal of the M-MuLV RT, and the chimeric polymerase expressed in silkworm larvae exhibited higher thermal stability and cDNA synthesis activity at 55 °C, a temperature at which the original M-MuLV RT loses its activity (Yano et al. 2019).

General procedure to generate chimeric DNA polymerase

Subsequent paragraphs detailed the standard for creating chimeric DNA polymerases with desired properties, suitable for a range of molecular biology applications to address specific challenges or enhance established techniques. The process follows the Design-Build-Test-Learn (DBTL) cycle, as depicted in the following steps (Fig. 5).

Fig. 5.

Fig. 5

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Schematic of general procedure for developing novel type of chimeric DNA polymerases. Applying the Design-Build-Test-Learn cycle inspired by synthetic biology, the procedure shown in the figure outlines the most significant steps in this procedure briefly

Design of the chimeric DNA polymerase

This section includes choosing parent polymerases, identifying related domains, and deciding in which way the domains will be connected. The first step is the selection of parent polymerases. Parent DNA polymerases with desired characteristics should be identified for subsequent integration. For example, if the goal is for enhanced thermal stability of the polymerase, it would be beneficial to explore DNA polymerase from thermophilic organisms. This approach is consistent with the principle of Form Follows Function (FFF). Besides, selected species that are closely related evolutionarily can increase the likelihood of successful recombination due to the higher homology between domains. The second step involves pinpointing the specific areas of interest within the proteins. DNA polymerases possess similar palm domains with necessary active sites, but their thumb and finger domains show quite different, potentially accounting for their distinct characteristics (Braithwaite and Ito 1993). It is crucial to determine which regions of the parent polymerases are responsible for the desired activity. You can opt for a rational design strategy on the basis of published literature, or, alternatively, employ a semi-rational design strategy with the help of computational models. The third step is the recombination design. This step is to decide how to integrate those domains in a manner that ensures they are organized in a structurally and functionally compatible way. Optional methods include tandem fusion, linker fusion, and domain insertion. Tandem fusion involves linking domains sequentially, either at N or C terminus. However, this method is not universally effective, given that the N- or C-termini of the peptide often play a pivotal role in achieving the desired protein conformation (Patel et al. 2022). Consequently, a linker is typically required to ensure adequate spacing between domains. Flexible or helical linkers, which are rich in small or hydrophilic amino acids, facilitate interactions between domains. For instance, the most widely used flexible linker is composed of a sequence of (Gly-Gly-Gly-Gly-Ser)n, and Gly-Thr-Gly-Ser-Gly-Thr or Gly-Ser-Gly-Gly-Val-Asp was used in aforementioned cases (Sun et al. 2006; Lee et al. 2013; Arai 2021). In contrast, rigid linker preserves the distance between domains through the use of helical structures or prolines residues. Typical motifs include the sequence (Glu-Ala-Ala-Ala-Lys)n and the pattern (aa-Pro)n, as summarized in a review (Patel et al. 2022). Domain insertion was carried out in a few cases (Bedford et al. 1997), probably due to the challenge of identifying a suitable ligation site within a domain.

Build up the chimeric DNA polymerase

The chimeric gene is synthesized using molecular biology techniques including restriction digestion and ligation, overlap extension PCR, and Gibson assembly. Then, it is cloned into an appropriate expression vector for expression and purification. The recombinant plasmid is transformed into suitable host cells, such as Escherichia coli or Pichia pastoris. Cells are cultured in an appropriate medium and polymerase protein is induced to express. Then, the chimeric DNA polymerase is purified from cell extracts using protein purification techniques, such as centrifugation, dialysis, and chromatography. Note that it may take several trials to decide the most efficient host and cloning method, but carefulness is always needed to maintain the activity of the polymerase as it is crucial for following characterization.

Test the characteristics of the chimeric DNA polymerase

The successful expression and purification of the chimeric DNA polymerase should be confirmed using techniques like SDS-PAGE and western blotting. The chimeric DNA polymerase can be characterized regarding its biochemical properties including activity, thermal stability, processivity, and fidelity. Briefly, the processivity of the polymerase is evaluated in single-stranded primer synthesis extension (Perumal 2023), and the activity of the DNA polymerase is monitored in double-stranded DNA synthesis with fluorescence dye (Yano et al. 2019) or radiolabeled nucleotide (Oscorbin et al. 2022). Thermal stability or resistance to inhibitors is tested by comparing the activity of the chimeric polymerase with the wild type in a gradient of temperature or some chemicals (Li et al. 2023). Note that different assay methods may result in contradicting conclusions as data might not be described in the same way (Oscorbin et al. 2017; Pavlov et al. 2012); thus, protocols and parameters, which may be found in the method section of published works in literature, should be confirmed carefully.

Learn from the result for debugging and optimization

If the initial design and construct fail to get the anticipated characteristics, check if there is anything wrong with the expression conditions that inactivated the chimeric DNA polymerase. Subsequent efforts involve altering some design, such as domain boundaries, linker sequences, homology of parent polymerases, or alleviating steric hindrance. Recent reports have also provided some novel points of view to deeply understand what makes a successful design, including analysis of charge distribution on the surface of the protein (Paik et al. 2022), analysis of the structure of the polymerase-DNA complex (Li et al. 2023), and the manipulation of conformation changes of the polymerase (Antony and Lohman 2019; Evans et al. 2022). Machine learning has been used to assist in evaluating the performance of the chimeras as well (Paik et al. 2023).

Challenges and perspectives

The numerous examples previously discussed have demonstrated the feasibility of optimizing DNA polymerases through the strategy of creating chimeric polymerases. However, there are still several challenges which are restricting the broader application of this approach.

Predicting the domains responsible for certain properties from parent polymerases can be challenging in the absence of a comprehensive understanding of the structure and the interactions between the domains. This endeavor necessitates insights from the field of structural biology, as well as the exploration of novel polymerases derived from organisms that inhabit extreme environments with high or low temperature, zero gravity, or strong radiation. Such efforts are crucial for further expanding the library of polymerase available for study (Dorawa et al. 2022; Ahlqvist et al. 2022).

Another important factor is the unpredictable interactions when various proteins or domains are fused with existing DNA polymerase. Identifying the most suitable chimeric polymerases from so many possibilities can be time-consuming and resource-intensive, especially when we lack the understanding of the factors that confer the desired characteristics. But there is an encouraging development that artificial intelligence (AI) powered by machine learning–based can prominently facilitate determining trials with the greatest potential by using computing models. It has an innate ability to uncover subtle relationships that may be imperceptible to human analysis, by processing vast amounts of data (Ferruz et al. 2023; Kouba et al. 2023).

It is also vital to consider the structural compatibility of the chimeric polymerase. The fusion of domains from different polymerases can lead to steric clashes or voids, potentially compromising the stability or activity of the chimeric polymerase to some extent. Hence, the design of a proper linker with optimal length and sequence to balance flexibility and rigidity of the chimera is highly significant (Patel et al. 2022; Huang et al. 2021). Moreover, charge engineering, which involves analyzing the effect of the surface charges on protein domains, may yield unexpected results (Paik et al. 2022; Martínez Del Río et al. 2023).

In some instances, fusing genes of the domains before translation, as mentioned earlier, may lead to detrimental domain interactions or reduced protein expression (Yu et al. 2015). To address this, a post-translational fusion strategy may be applied. This post-translational conjugation technique by fusing protein after ribosomal translation has also been reported as effective in some cases, which was summarized in a recent review by Taylor et al. (2022). Although this approach has not yet been tested with DNA polymerases, it presents an intriguing opportunity and is worth a try.

Despite facing these challenges, progress in the fields of protein engineering, structural biology, molecular evolution, and AI has empowered researchers to surmount these obstacles and develop chimeric DNA polymerases that exhibit innovative characteristics and improved performance. In conclusion, the ongoing research in these areas is anticipated to deepen our understanding of DNA polymerases. Consequently, this intensive knowledge will lay the groundwork for a more extensive spectrum of applications across diverse domains.

Author contribution

ZY and JW designed, wrote, and approved the manuscript.

Funding

This research was funded by the Science and Technology Program of Guangzhou (2024B01J1277, 202205110007).

Declarations

Ethics approval

This article does not contain any studies with human participants performed by any of the authors.

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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